Nucleic acid enzyme biosensors for ions

ABSTRACT

Disclosed are compositions and methods for the sensitive and selective detection of ions using nucleic acid enzymes.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0001] The U.S. Government may have rights in the present inventionpursuant to the terms of grant number R29 GM53706 awarded by theNational Institutes of Health.

BACKGROUND

[0002] Many metals pose a risk as environmental contaminants. Awell-known example is lead. Low level lead exposure can lead to a numberof adverse health effects, with as many as 9-25% of pre-school childrenpresently at risk. The level of lead in the blood considered toxic is≧10 μg/dL (480 nM). Current methods for lead analysis, such as atomicabsorption spectrometry, inductively coupled plasma mass spectrometry,and anodic stripping voltammetry, often require sophisticated equipment,sample pre-treatment, and skilled operators.

[0003] Simple, rapid, inexpensive, selective and sensitive methods thatpermit real time detection of Pb²⁺ and other metal ions are veryimportant in the fields of environmental monitoring, clinicaltoxicology, wastewater treatment, and industrial process monitoring.Furthermore, methods are needed for monitoring free or bioavailable,instead of total, metal ions in industrial and biological systems.

[0004] Fluorescence spectroscopy is a technique well suited for verysmall concentrations of analytes. Fluorescence provides significantsignal amplification, since a single fluorophore can absorb and emitmany photons, leading to strong signals even at very low concentrations.In addition, the fluorescence time-scale is fast enough to allowreal-time monitoring of concentration fluctuations. The fluorescentproperties only respond to changes related to the fluorophore, andtherefore can be highly selective. Furthermore, fluorometers for uses inthe field are commercially available. Fluorescent detection is alsocompatible with fiber-optic technology and well suited for in vivoimaging applications. Several fluorescence-related parameters can beassessed for the purpose of sensing, including fluorescence intensity,emission or excitation wavelength, fluorescence lifetime and anisotropy.

[0005] Many fluorescent chemosensors, including fluorophore-labeledorganic chelators (Rurack, et al., 2000; Hennrich et al., 1999; Winkleret al., 1998; Oehme & Wolfbeis, 1997) and peptides (Walkup & Imperiali,1996; Deo & Godwin, 2000; Pearce et al., 1998), have been developed formetal ion detection. These ion sensors are usually composed of anion-binding motif and a fluorophore. Metal detection using thesefluorescent chemosensors relies on the modulation of the fluorescentproperties of the fluorophore by the metal-binding event. Detectionlimits on the level of micromolar and even nanomolar concentrations havebeen achieved for heavy metal ions including Zn²⁺, Cu⁺, Hg²⁺, Cd²⁺ andAg⁺. The design and synthesis of a chemosensor that exhibits highlyselective and sensitive binding of the metal ion of choice in aqueoussolution is still a big challenge, although the metal binding and thefluorescent moieties of the sensor can be systematically varied toachieve desired properties.

[0006] Nucleic acid molecules have previously been adapted to sense thepresence of nucleic acids and to detect gene mutations from inheriteddiseases or chemical damages. In recent years, the molecular recognitionand catalytic function of nucleic acids have been extensively explored.This exploration has lead to the development of aptamers and nucleicacid enzymes.

[0007] Aptamers are single-stranded oligonucleotides derived from an invitro evolution protocol called systematic evolution of ligands byexponential enrichment (SELEX). Nucleic acid aptamers have been isolatedfrom random sequence pools and can selectively bind to non-nucleic acidtargets, such as small organic molecules or proteins, with affinities ashigh as 10⁻¹⁴ M (Uphoffet al., 1996; Famulok, 1999). Most aptamersundergo a conformational change when binding their cognate ligands. Withthis property, several DNA and RNA aptamers have been engineered tosense L-adenosine or thrombin through an internally labeled fluorescentreporter group (Jhaveri et al., 2000). Thus, the conformational changein the aptamer upon binding leads to a change in fluorescence.

[0008] Nucleic acid enzymes are nucleic acid molecules that catalyze achemical reaction. In vitro selection of nucleic acid enzymes from alibrary of 10¹⁴-10¹⁵ random nucleic acid sequences offers considerableopportunity for developing enzymes with desired characteristics (Breaker& Joyce, 1994; Breaker, 1997). Compared with combinatorial searches ofchemo- and peptidyl-sensors, in vitro selection of DNA/RNA is capable ofsampling a larger pool of sequences, amplifying the desired sequences bypolymerase chain reactions (PCR), and introducing mutations to improveperformance by mutagenic PCR.

[0009] Allosteric ribozymes (or aptazymes), which combine the featuresof both aptamer and catalytic RNA, also hold promises for sensing smallmolecules (Potyrailo et al., 1998; Koizumi et al., 1999; Robertson &Ellington, 1999, 2000). Their reactivity is modulated through theconformational changes caused by the binding of small organic moleculesto an allosteric aptamer domain. Therefore, the signal of ligand bindingcan be transformed into a signal related to chemical reaction.

[0010] Divalent metal ions can be considered as a special class ofcofactors controlling the activity of nucleic acid enzymes. The reactionrate of the nucleic acid enzymes depends on the type and concentrationof the metal ion in solution. Several RNA and DNA enzymes obtainedthrough in vitro selection are highly specific for Cu²⁺, Zn²⁺, and Pb²⁺,with metal ion requirements on the level of micromolar concentrations(Breaker & Joyce, 1994; Pan & Uhlenbeck, 1992; Carmi et al., 1996; Panet al., 1994; Cuenoud & Szotak, 1995; Li et al., 2000; Santoro et al.,2000).

BRIEF SUMMARY

[0011] The present invention uses nucleic acid enzymes as signaltransducers for ion detection. Compared with fluorescent chemosensor andprotein biosensors, nucleic acid-based sensors are more amenable tocombinatorial search for sequences with desired metal specificity andaffinity. In addition, DNA, in particular, is stable and can be readilysynthesized. A wide range of fluorescent dyes can be easily introducedat specific sites to suit different needs. DNA-based biosensors can alsobe adapted for use with optical fiber and DNA-chip technology forapplications such as in vivo imaging, in situ detection, and arraysensing.

[0012] In one aspect, the present invention provides for specific andsensitive biosensors of ions. The biosensors are useful in methods ofdetecting the presence of an ion, particularly metal ions such as Pb²⁺.In certain embodiments, the biosensors may be used to determine theconcentration of a particular ion in a solution.

[0013] The biosensors of the present invention use nucleic acid enzymesthat require the presence of specific ions for their activity. Enzymaticactivity leads to hydrolytic cleavage of a substrate nucleic acid thatmay be part of the nucleic acid enzyme itself. The resulting cleavageproduct then may be detected indicating the presence of the ion.

[0014] In a preferred embodiment, the biosensor comprises a fluorophoreand a quencher arranged in proximity such that prior to cleavage thefluorescence intensity is decreased by the quencher. However, uponcleavage, the fluorophore and quencher are separated leading to anincrease in fluorescence intensity. In a further preferred embodiment,the biosensor contains an array of nucleic acid enzymes having a rangeof sensitivities and specificities to several different ions.

[0015] A “nucleic acid enzyme” is a nucleic acid molecule that catalyzesa chemical reaction. The nucleic acid enzyme may be covalently linkedwith one or more other molecules yet remain a nucleic acid enzyme.Examples of other molecules include dyes, quenchers, proteins, and solidsupports. The nucleic acid enzyme may be entirely made up ofribonucleotides, deoxyribonucleotides, or a combination of ribo- anddoxyribonucleotides.

[0016] A “sample” may be any solution that may contain an ion (before orafter pre-treatment). The sample may contain an unknown concentration ofan ion. For example, the sample may be paint that is tested for leadcontent. The sample may be diluted yet still remains a sample. Thesample may be obtained from the natural environment, such as a lake,pond, or ocean, an industrial environment, such as a pool or wastestream, a research lab, common household, or a biological environment,such as blood. Of course, sample is not limited to the taking of analiquot of solution but also includes the solution itself. For example,a biosensor may be placed into a body of water to measure forcontaminants. In such instance, the sample may comprise the body ofwater or a particular area of the body of water. Alternatively, asolution may be flowed over the biosensor without an aliquot beingtaken. Furthermore, the sample may contain a solid or be produced bydissolving a solid to produce a solution. For example, the solution maycontain soil from weapon sites or chemical plants.

[0017] “Measuring the product of the nucleic acid enzymatic reaction”includes measuring the result of the production of a product by anenzyme. For example, in an embodiment where the substrate comprises aquencher and the enzyme comprises a fluorophore and cleavage of thesubstrate by the enzyme leads to dissociation of the product from theenzyme, “measuring the product” includes detecting the increase offluorescence. Thus, one is measuring the product by detecting itsinability to quench fluorescence.

[0018] “Contacting a nucleic acid enzyme with a sample” includes placingthe sample and enzyme in proximity such that an ion in the sample couldbe used as a cofactor. “Contacting” includes such acts as pipetting asample onto a solid support or into a tube or well containing thenucleic acid enzyme. Alternatively, the enzyme may be brought to thesample. For example, the enzyme may be placed into a stream to monitorfor the presence of a contaminant.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019]FIG. 1. Selection scheme for RNA-cleaving deoxyribozymes. FIG. 1A.Starting pool of random-sequenced DNAs, engineered to contain twosubstrate-binding domains. Each member of the pool contains a5′-terminal biotin (encircled B), a single embedded ribonucleotide (rA)and a 40-nucleotide random sequence domain (N40). FIG. 1B. Selectiveamplification scheme for isolation of DNA that catalyzes the metalcofactor (Co²⁺ or Zn²⁺) dependent cleavage of an RNA phosphodiester.

[0020]FIG. 2. Sequence classes of the cloned Zn-DNA. The numbers on theleft are the clone-numbers randomly assigned to the sequences during thecloning and sequencing process. The highly conserved sequences(Region-20nt) are in bold. The covariant nucleotides are underlined. The5′- and the 3′-primer binding sequences are shown in italic.

[0021]FIG. 3. Sequence classes of the cloned Co-DNA. The clone-numbersare listed on the left. The 5′ and the 3′ primer binding sequences arein italic.

[0022]FIG. 4. Sequence alignment of the N40 region of the reselectedZn-DNAs. The wild-type sequence is listed on the top, followed by thereselected Zn-DNA sequences. Only the point mutations are shown for thereselected sequences, while the nucleotides that are identical to thewild type at the corresponding positions are omitted. Numbers listed onthe left are clone-numbers. The rate constants (k_(obs)) of severalreselected Zn-DNA in 100 μM Zn²⁺ are shown on the right.

[0023]FIG. 5. Proposed secondary structure of the Zn(II)-dependenttrans-cleaving deoxyribozyme.

[0024]FIG. 6. Sequences and proposed secondary-structures of severalRNA-cleaving deoxyribozymes. FIG. 6A and FIG. 6B. The deoxyribozymeselected using Mg²⁺ or Pb²⁺ as cofactor (Breaker & Joyce, 1994, 1995).FIG. 6C and FIG. 6D. The 10-23 and the 8-17 deoxyribozymes selected inMg²⁺ to cleave all-RNA substrate (Santoro & Joyce, 1997). FIG. 6E. Adeoxyribozyme selected using L-histidine as cofactor. FIG. 6F. The 17Edeoxyribozyme selected in Zn²⁺. In each structure, the upper strand isthe substrate and the lower strand is the enzyme. Arrows identify thesite of RNA transesterification.

[0025]FIG. 7. Comparison of G3 deoxyribozyme with class II Co-DNA. FIG.7A. The predicted secondary structure of the G3 deoxyribozyme (Geyer &Sen, 1997). X represents variable sequences. The boxed region was alsofound in class II Co-DNA FIG. 7B. The minimal structure motif of theclass II Co-DNA predicted by mfold program. The arrows indicate thecleavage sites.

[0026]FIG. 8. Steady-state fluorescence spectra of the substrate(Rh-17DS) alone (I), after annealing to the deoxyribozyme (17E-Dy) (II),and 15 min after adding 500 nM Pb(OAc)₂ (III).

[0027]FIG. 9. The fluorescence response rate (vfluo) of Rh-17EDS-Dy fordifferent divalent metal ions. The “control” was measured without Pb(II)or transition metal ions. FIG. 9A. with 500 nM M(II) in 50 mM HEPES (pH7.5); The inserted graph shows the change of fluorescence intensity at580 nm in response to the addition of M(II). The curve with dramaticchange was collected in Pb(II), the other curves were collected in oneof the other eight divalent metal ions. FIG. 9B. with 500 nM M(II) in100 mM NaCl, 1 mM Mg²⁺, 1 mM Ca²⁺ and 50 mM HEPES (pH 7.5).

[0028]FIG. 10. Dependence of v_(fluo) on the concentration of Pb 2+ orCO²⁺. The reaction was carried out in the presence of 50 mM NaCl in 50mM HEPES (pH 7.5). FIG. 10A. The initial rate (v_(fluo)) increased withthe concentration of Pb²⁺ (♦) and Co²⁺ (▪) over a range of three ordersof magnitude. FIG 10B. At low concentrations, V_(fluo) increasedlinearly with Pb²⁺ (♦) or Co²⁺ (▪) concentration.

[0029]FIG. 11. DNA chips for ion sensing. FIG. 11A. The array ofdeoxyribozymes with different metal specificity and affinity on the DNAchip for metal ion sensing. FIG. 11B. Quantitative and qualitativedetection of metal ions using the metal ion-sensing deoxyribozyme chip.The z-axis represents the fluorescence intensity change upon theexposure of the chip to the sample under examination. The change in thefluorescence intensity is caused by the deoxyribozyme-catalyzedsubstrate cleavage in the presence of a specific kind and concentrationof metal ion.

DETAILED DESCRIPTION

[0030] The invention described herein represents a new class of ionsensors and is the first example of a DNA enzyme-based biosensor forions. It combines the high selectivity of DNA enzymes with the highsensitivity of fluorescence detection. For example, in one embodiment,selectivity for Pb²⁺ was >80 fold over other divalent metal ions withhigh sensitivity (>400% signal increase). Such selectivity andsensitivity provides for qualitative and quantitative detection of ionsover a concentration range of several orders of magnitude. In apreferred embodiment, the fluorescence domain is decoupled from theion-recognition/catalysis domain, and therefore the sensitivity andselectivity of this system may be manipulated by a careful choice offluorophores and by performing in vitro selection of ion-binding domainsto not only keep sequences reactive with the ion of choice, but alsoremove sequences that also respond to other ions.

[0031] In addition, DNA is stable, inexpensive and easily adaptable tooptical fiber and chip technology for device manufacture. The attachmentof DNA enzymes to optical fibers or chips allows regeneration of thesensors by washing away the cleavage products and adding new substrates.Finally, sequences specific for other ions and with various detectionranges may be isolated by varying the selection conditions, providingfor a highly sensitive and selective fluorosensor system.

[0032] Nucleic Acid Enzymes

[0033] A growing number of nucleic acid enzymes have been discovered ordeveloped showing a great diversity in catalytic activity (Table 1 andTable 2). Many if not all of the enzymes are dependent on one or moreion cofactors. In vitro selection may be used to “enhance” selectivityand sensitivity for a particular ion. Such enzymes find particularutility in the compositions and methods of the present invention. Forexample, nucleic acid enzymes that catalyze molecular association(ligation, phophorylation, and amide bond formation) or dissociation(cleavage or transfer) are particularly useful.

[0034] In preferred embodiments, a nucleic acid enzyme that catalyzesthe cleavage of a nucleic acid in the presence of an ion is used. Thenucleic acid enzyme may be RNA (ribozyme), DNA (deoxyribozyme), aDNA/RNA hybrid enzyme, or a peptide nucleic acid (PNA) enzyme. PNAscomprise a polyamide backbone and the bases found in naturally occurringnucleosides and are commercially available from, e.g., Biosearch, Inc.(Bedford, Mass.).

[0035] Ribozymes that may be used in the present invention include, butare not limited to, group I and group II introns, the RNA component ofthe bacterial ribonuclease P, hammerhead, hairpin, hepatitis delta virusand Neurospora VS ribozymes. Also included are in vitro selectedribozymes, such as those isolated by Tang and Breaker (2000).

[0036] One limitation of using a ribozyme is that they tend to be lessstable than deoxyribozymes. Thus, in preferred embodiments, the nucleicacid enzyme is a deoxyribozyme. Preferred deoxyribozymes include thoseshown in FIG. 6A-6F and deoxyribozymes with extended chemicalfunctionality (Santoro et al., 2000). TABLE 1 Reactions catalyzed byribozymes that were isolated from in vitro selection experiments.Reaction k_(cat) (min⁻¹) k_(m) (μM) k_(cat)/k_(uncat) ^(a) ReferencePhosphoester centers Cleavage 0.1 0.03 10⁵ Vaish, 1998 Transfer 0.3 0.02 ¹⁰ ¹³ Tsang, 1996 Ligation 100 9 10⁹ Ekland, 1995 Phosphorylation 0.340 >10⁵  Lorsch, 1994 Mononucleotide 0.3 5000 >10⁷  Ekland, 1996polymerization Carbon centers Aminoacylation 1 9000 10⁶ Illangasekare,1997 Aminoacyl ester 0.02 0.5 10 Piccirilli, 1992 hydrolysis Aminoacyltransfer 0.2 0.05 10³ Lohse, 1996 N-alkylation 0.6 1000 10⁷ Wilson, 1995S-alkylation 4 × 10⁻³ 370 10³ Wecker, 1996 Amide bond 1 × 10⁻⁵ 10² Dai,1995 cleavage Amide bond 0.04 2 10⁵ Wiegand, formation 1997 Peptide bond0.05 200 10⁶ Zhang, 1997 formation Diels-Alder >0.1 >500 10³ Tarasow,1997 cycloaddition Others Biphenyl 3 × 10⁻⁵ 500 10² Prudent, 1994isomerization Porphyrin 0.9 10 10³ Conn, 1996 metallation

[0037] TABLE 2 Deoxyribozymes isolated through in vitro selection.Reaction Cofactor k_(max)(min⁻¹)^(a) k_(cat)/k_(uncat) Reference RNAPb²⁺ 1 10⁵ Breaker, 1994 transesterification Mg²⁺ 0.01 10⁵ Breaker, 1995Ca²⁺ 0.08 10⁵ Faulhammer, 1997 Mg²⁺ 10 >10⁵  Santoro, 1997 None 0.01 10⁸Geyer, 1997 L-histidine 0.2 10⁶ Roth, 1998 Zn²⁺ ˜40 >10⁵  Li, J., 2000DNA cleavage Cu²⁺ 0.2 >10⁶  Carmi, 1996 DNA ligation Cu²⁺ or 0.07 10⁵Cuenod, 1995 Zn²⁺ DNA Ca²⁺ 0.01 10⁹ Li, Y., 1999 phosphorylation 5′,5′-Cu²⁺ 5 × 10⁻³  >10¹⁰  Li, Y., 2000 pyrophophate formation Porphyrin None1.3 10³ Li, Y., 1996 metalation

[0038] An advantage of ribozymes and deoxyribozymes is that they may beproduced and reproduced using biological enzymes and appropriatetemplates. However, the present invention is not limited to ribozymesand deoxyribozymes. Nucleic acid enzymes that are produced by chemicaloligosynthesis methods are also included. Thus, nucleic acids includingnucleotides containing modified bases, phosphate, or sugars may be usedin the compositions and methods of the present invention. Modified basesare well known in the art and include inosine, nebularine, 2-aminopurineriboside, N⁷-denzaadenosine, and O⁶-methylguanosine (Earnshaw & Gait,1998). Modified sugars and phosphates are also well known and include2′-deoxynucleoside, abasic, propyl, phosphorothioate, and 2′-O-allylnucleoside (Earnshaw & Gait, 1998). DNA/RNA hybrids and PNAs may be usedin the compositions and methods of the present invention. The stabilityof PNAs and relative resistance to cellular nucleases make PNA enzymesamenable to in vivo applications.

[0039] In certain embodiments, the substrate for the nucleic acid enzymeand the enzyme itself are contained in the same nucleic acid strand.Such enzymes are cis-acting enzymes. Examples include the Zn²⁺-dependentdeoxyribozymes (Zn-DNA) created in Example 1 (FIG. 1A and FIG. 2).

[0040] In preferred embodiments, the nucleic acid enzyme cleaves anucleic acid strand that is separate from the strand comprising theenzyme (trans-acting). One advantage of utilizing trans-activity isthat, after cleavage, the product is removed and additional substratemay be cleaved by the enzymatic strand. A preferred nucleic acid enzymeis 5′-CATCTCTTCTCCGAGCCGGTCGAAATAGTGAGT-3′ (17E; FIG. 5; SEQ ID NO:1).The corresponding preferred substrate to 17E is5′-ACTCACTATrAGGAAGAGATG-3′ (17DS; FIG. 5; SEQ ID NO:2), where rAdenotes a single ribonucleotide.

[0041] It may be beneficial to use directed mutation to change one ormore properties of a nucleic acid enzyme or its substrate. Using 17E and17DS as an example, one may wish to alter the avidity of the two arms ofthe hybridized enzyme and substrate. The “arms” are those areasdisplaying Watson-Crick basepairing in FIG. 5. To alter avidity, one mayincrease or decrease the length of the arms. Increasing the length ofthe arms increases the number of Watson-Crick bonds, thus increasing theavidity. The opposite is true for decreasing the length of the arms.Decreasing the avidity of the arms facilitates the removal of substratefrom the enzyme, thus allowing faster enzymatic turnover.

[0042] Another method of decreasing avidity includes creating mismatchesbetween the enzyme and the substrate. Alternatively, the G-C content ofthe arms may be altered. Of course, the effect of any directed changeshould be monitored to ensure that the enzyme retains its desiredactivity, including ion sensitivity and selectivity. In light of thepresent disclosure, one of skill in the art would understand how tomonitor for a desired enzymatic activity. For example, to ensure thatthe mutated enzyme maintained sensitivity and selectivity for Pb²⁺, onewould test to determine if the mutated enzyme remained reactive in thepresence of lead (sensitivity) and maintained its lower level ofactivity in the presence of other ions (selectivity).

[0043] In preferred embodiments, the nucleic acid enzyme is sensitiveand selective for a single ion. The ion may be any anion or cation. Theion may be monovalent, divalent, trivalent, or polyvalent. Examples ofmonovalent cations include K⁺, Na⁺, Li⁺, Tl⁺, NH₄ ⁺, and Ag⁺. Examplesof divalent cations include Mg²⁺, Ca²⁺, Mn²⁺, Co²⁺, Ni²⁺, Zn²⁺, Cd²⁺,Cu²⁺, Pb²⁺, Hg²⁺, Pt²⁺, Ra²⁺, Ba²⁺, and Sr²⁺. Examples of trivalentcations include Co³⁺, Cr³⁺, and lanthanide ions (Ln³⁺). Polyvalentcations include Ce⁴⁺, spermine, and spermidine. Because, in certainembodiments, the biosensors of the present invention are used to monitorcontaminants in the environment, preferred ions are those that are toxicto living organisms, e.g., Ag⁺, Pb²⁺ and Hg²⁺.

[0044] Often the nucleic acid enzymes that have activity with one ionalso have at least some activity with one or more other ions. Suchmulti-sensitive enzymes may stilt be used in the compositions andmethods of the present invention. However, it should be understood thatuse of a multi-sensitive enzyme may lead to uncertainty as to which ofthe ions is present. In such cases, measuring the rate of enzymaticactivity, using serial dilutions, or using an array of nucleic acidenzymes may be helpful in deciphering which ion is present.

[0045] In Vitro Selection of Nucleic Acid Enzymes

[0046] Many nucleic acid enzymes that are dependent on ions,particularly metal ions, for activity are known in the art (Breaker &Joyce, 1994; Pan & Uhlenbeck, 1992; Cuenoud & Szostak, 1995; Carmi etal., 1996; Li et al., 2000; Santoro et al., 2000). In light of thepresent disclosure, one of skill in the art would understand how toutilize a known nucleic acid enzyme in the methods and biosensors of thepresent invention. Furthermore, the present invention may include anucleic acid enzyme created by in vitro selection. Methods of in vitroselection of nucleic acid enzymes are known in the art and describedherein.

[0047] In vitro selection is a technique in which RNA or DNA moleculeswith certain functions are isolated from a large number of sequencevariants through multiple cycles of selection and amplification (Joyce,1994; Chapman et al., 1994). The concept of in vitro selection ofcatalytic RNA molecules was first introduced in the late 1980's. Sincethen, it has been widely applied to obtain ribozymes with maximizedactivities or novel catalytic abilities, and to identifyoligonucleotides (called aptamers) that bind to certain proteins orsmall molecules with high affinity. The process for aptamers selectionis sometimes referred as systematic evolution of ligands by exponentialenrichment (SELEX)(Tuerk & Gold, 1990).

[0048] The first catalytic DNA (deoxyribozyme) was isolated by Breakerand Joyce in 1994 through in vitro selection. This deoxyribozyme is ableto catalyze phosphodiester cleavage reaction in the presence of Pb²⁺.Unlike RNA-based catalysts, DNA molecules with catalytic functions havenot been encountered in nature, where DNA exists primarily asbase-paired duplex and serves mainly as the carrier of geneticinformation. The identification of DNA molecules with catalyticfunctions further demonstrated the power of in vitro selection.

[0049] In vitro selection is typically initiated with a large collectionof randomized sequences. A typical DNA or RNA library for selectioncontains 10¹³-10¹⁶ sequence variants. The construction of a completelyrandomized pool is accomplished by chemical synthesis of a set ofdegenerated oligonucleotides using standard phosphoramidite chemistry.The 3′-phosphoramidite compounds of four nucleosides (A, C, G, and T)are premixed before being supplied to an automated DNA synthesizer toproduce oligonucleotides. By controlling the ratio of fourphosphoroamidites, the identity at each nucleotide position can beeither completely random, i.e. with equal chance for each base, orbiased toward a single base. Other strategies for creating a randomizedDNA library include applying mutagenic polymerase chain reaction (PCR)and template-directed mutagenesis (Tsang and Joyce, 1996; Cadwell andJoyce, 1992, 1994). For the purpose of in vitro selection of functionalRNA molecules, the randomized DNA library is converted to an RNA librarythrough in vitro transcription.

[0050] In vitro selection takes advantage of a unique property of RNAand DNA, i.e., the same molecule can possess both genotype (codinginformation) and phenotype (encoded function). The DNA or RNA moleculesin the randomized library are screened simultaneously. Those sequencesthat exhibit a desired function (phenotype) are separated from theinactive molecules. Usually the separation is performed through affinitycolumn chromatography, being linked to or released from a solid support,gel electrophoresis separation, or selective amplification of a taggedreaction intermediate. The genotype of the active molecules are thencopied and amplified, normally through polymerase chain reaction (PCR)for DNA or isothermal amplification reaction for RNA (Guatelli et al.,1990). Mutations can be performed with mutagenic PCR to reintroducediversity to the evolving system. These three steps—selection,amplification and mutation, are repeated, often with increasingselection stringency, until sequences with the desired activity dominatethe pool.

[0051] Novel nucleic acid enzymes isolated from random sequences invitro have extended the catalytic repertoire of RNA and DNA far beyondwhat has been found in nature. The selected ribozymes are capable ofcatalyzing a wide range of reactions at both phosphate and non-phosphatecenters (Table 1). The reactions that are catalyzed by deoxyribozymesare less diverse, compared with the ribozymes (Table 2). However, thecatalytic rate (k_(cat)) of most deoxyribozymes is comparable to that ofthe ribozymes catalyzing the same reaction. In certain cases, thecatalytic efficiency (k_(cat)/K_(m)) of nucleic acid enzymes evenexceeds that of the protein enzymes.

[0052] In vitro selection can be used to change the ion specificity orbinding affinity of existing ribozymes, or to obtain nucleic acidenzymes specific for desired ions. For example, in vitro-selectedvariants of the group I intron (Lehman & Joyce, 1993) and the RNase Pribozyme (Frank & Pace, 1997) have greatly improved activity in Ca²⁺,which is not an active metal ion cofactor for native ribozymes. The Mg²⁺concentration required for optimal hammerhead ribozyme activity has beenlowered using in vitro selection to improve the enzyme performance underphysiological conditions (Conaty et al., 1999; Zillman et al., 1997).Breaker and Joyce have isolated several RNA-cleaving deoxyribozymesusing Mg²⁺, Mn²⁺, Zn²⁺, or Pb²⁺ as the cofactor (Breaker & Joyce, 1994,1995). Only the sequence and structure of the Pb²⁺-dependent and theMg²⁺-dependent deoxyribozymes were reported (FIGS. 6A and 6B). Otherexamples of metal-specific RNA/DNA enzymes obtained through in vitroselection include a Pb²⁺-specific RNA-cleaving ribozyme (calledleadzyme) (Pan & Uhlenbeck, 1992), a Cu²⁺-specific DNA-cleavingdeoxyribozyme (Carmi et al., 1996), and a DNA ligase active in Zn²⁺ andCu²⁺ (Cuonod & Szostak, 1995).

[0053] Often nucleic acid enzymes developed for a specific metal ion byin vitro selection will have activity in the presence of other metalions. For example, 17E deoxyribozyme was developed by in vitro selectionfor activity in the presence of Zn²⁺. Surprisingly, the enzyme showedgreater activity in the presence of Pb²⁺ than Zn²⁺. Thus, althoughproduced in a process looking for Zn²⁺-related activity, 17E may be usedas a sensitive and selective sensor of Pb²⁺.

[0054] To produce nucleic acid enzymes with greater selectivity, anegative selection step may be included in the process. For Example,Pb²⁺-specific deoxyribozymes may be isolated using a similar selectionscheme as for the selection of Co²⁺- and Zn²⁺-dependent DNA enzymesdescribed in Example 1. In order to obtain deoxyribozymes with highspecificity for Pb²⁺, negative-selections may be carried out in additionto the positive selections in the presence of Pb²⁺.

[0055] For negative selection, the DNA pool is selected against a “metalsoup”, which contains various divalent metal ions (e.g. Mg²⁺, Ca²⁺,Mn²⁺, Zn²⁺, Cd²⁺, Co²⁺, Cu²⁺, etc.). Those sequences that undergoself-cleavage in the presence of divalent metal ions other than Pb²⁺ arethen washed off the column. The remaining sequences are further selectedwith Pb²⁺ as the cofactor. Pb²⁺-dependent deoxyribozymes with differentaffinities for Pb²⁺ can be obtained by controlling the reactionstringency (Pb²⁺ concentration).

[0056] Fluorophores and Quenchers

[0057] Any chemical reaction that leads to a fluorescent orchemiluminescent signal may be used in the compositions and methods ofthe present invention. In preferred embodiments, fluorophores are usedto measure enzymatic activity and, thus, detect the presence of aparticular ion. Essentially any fluorophore may be used, includingBODIPY, fluoroscein, fluoroscein substitutes (Alexa Fluor dye, Oregongreen dye), long wavelength dyes, and UV-excited fluorophores. These andadditional fluorphores are listed in Fluorescent and Luminescent Probesfor Biological Activity. A Practical Guide to Technology forQuantitative Real-Time Analysis, Second Ed. W. T. Mason, ed. AcademicPress (1999) (incorporated herein by reference). In preferredembodiments, the fluorophore is 6-carboxytetramethylrhodamin (TAMRA).TAMRA has an excitation range of 500-550 nm and an emission range of560-650 nm.

[0058] In certain embodiments, the substrate is labeled with afluorophore and measurement of enzymatic activity is done by detectingthe non-hybridized cleavage products in solution. Preferably, this isdone by measuring the level of fluorescence in solution withoutdetecting fluorescence from the bound substrate. This may be done bycreating a flow such that, once the cleavage product enters thesolution, it is carried away by the flow. Fluorescence of the flow isthen measured in an area away from the enzyme-substrate pairs.

[0059] In preferred embodiments, the substrate is labeled with afluorophore but fluorescence is quenched by a nearby quenching molecule.Quenching molecules absorb the energy of the excited fluorophore. Closeproximity of fluorophore and quencher allow for the energy to betransferred from the fluorophore to the quencher. By absorbing thisenergy, the quencher prevents the fluorophore from releasing the energyin the form of a photon.

[0060] Quenchers may be categorized as non-fluorescent and fluorescentquenchers. Non-fluorescent quenchers are capable of quenching thefluorescence of a wide variety of fluorophores. Generally,non-fluorescent quenchers absorb energy from the fluorophore and releasethe energy as heat. Examples of non-fluorescent quenchers includeDABCYL, QSY-7, and QSY-33.

[0061] Fluorescent quenchers tend to be specific to fluorophores thatemit at a specific wavelength range. Fluorescent quenchers often involvefluorescence resonance energy transfer (FRET). In many instances thesecond molecule is also a fluorophore. In such cases, close proximity ofthe fluorophore and quencher is indicated by a decrease in fluorescenceof the “fluorophore” and an increase in fluorescence in the fluorescentquencher. Commonly used fluorescent fluorophore pairs(fluorophore/fluorescent quencher) includefluorescein/tetramethylrhodamine, IAEDANS/fluorescein,fluorescein/fluorescein, and BODIPY FL/BODIPY FL.

[0062] The quencher may be located on a support such that it is inproximity with the fluorophore when the substrate is bound to theenzyme. In preferred embodiments, the quencher is linked to the enzyme.Even more preferred is to have the fluorphore linked to the 5′ end ofthe substrate and the quencher linked to the 3′ end of the enzyme suchthat when the substrate and enzyme are hybridized, the fluorophore andthe quencher are in close proximity to each other. Upon cleavage of thesubstrate, the product disassociates from the enzyme. Dissociationremoves the fluorophore from the quencher, leading to an increase influorescence (FIG. 8).

[0063] Of course, it would be understood that the fluorophore could belinked essentially anywhere on the substrate and quencher essentiallyanywhere on the enzyme, as long as they are in close proximity to eachother when the enzyme is hybridized to the substrate. By closeproximity, it is meant that they are situated such that the quencher isable to function. Furthermore, the quencher may be placed on thesubstrate and the fluorophore on the enzyme. Alternatively, bothquencher and fluorophore may be linked to the substrate on opposite endsfrom the potential cleavage site. Cleavage of such a molecule would leadto dissociation of the two ends and thus separation of the fluorophoreand quencher, leading to an increase in fluorescence. Similarly, inembodiments wherein the enzyme and the substrate are contained withinthe same nucleic acid strand, the fluorophore and quencher may be placedon opposite ends of the cleavage site.

[0064] When choosing a fluorophore, quencher, or where to position themolecules, it is important to consider, and preferably to test, theeffect of the fluorphore or quencher on the enzymatic activity of thenucleic acid enzyme. Also, it is preferable that the fluorophore displaya high quantum yield and energy transfer efficiency. Long-wavelength(excitation and emission) fluorophores are preferred because of lessinterference from other absorbing species. The fluorophore should alsobe less sensitive to pH change or to non-specific quenching by metalions or other species.

[0065] Methods and devices for detecting fluorescence are welldeveloped. Essentially any instrument or method for detectingfluorescent emissions may be used. For example, WO 99/27351(incorporated herein in its entirety) describes a monolithicbioelectronical device comprising a bioreporter and an opticalapplication specific integrated circuit (OASIC). The device allowsremote sampling for the presence of substances in solution.

[0066] Furthermore, the fluorescence may be measured by a number ofdifferent modes. Examples include fluorescence intensity, lifetime, andanisotropy in either steady state or kinetic rate change modes(Lakowicz, 1999).

[0067] Sometimes other factors in a solution such as pH, saltconcentration or ionic strength, or viscosity will have an effect onfluorescence. Others may affect the hybridization of the substrate andenzyme. Therefore, in preferred methods, controls are run to determineif the solution itself, regardless of enzymatic activity, is alteringthe fluorescence. Such controls include the use of non-cleavablesubstrates and or substrate without the presence of enzyme.

[0068] Biosensors

[0069] Described herein are nucleic acid enzymes that are dependent onthe presence of a specific ion for activity. Using fluorophores orfluorophore/quencher labeling, it is possible to measure enzymaticactivity, even in real time. These qualities make the compositions ofthe present invention excellent for use in biosensors for detectingions.

[0070] Many biosensors utilizing nucleic acids are known in the art. Forexample, biosensors using aptamers have been developed for detectingmolecules such as thrombin or adenosine (Potyrailo et al., 1999; Lee &Walt, 2000). In light of the present disclosure, one of ordinary skillin the art would know how to modify the nucleic acid biosensors toinclude nucleic acid enzymes.

[0071] In a simple embodiment, a biosensor of the present inventioncomprises a nucleic acid enzyme labeled with a quencher, a substratelabeled with a fluorophore, and a device to detect fluorescence such asa fluorescence microscope or a fluorometer. In a method using thisembodiment, the enzyme and substrate are contacted with a samplesuspected of containing an ion to which the enzyme is sensitive.Fluorescence is measured and compared to a control wherein the ion isabsent. Change in fluorescence is indicative of the presence of the ion.

[0072] Of course, many variants of even this simple embodiment areincluded within the scope of the invention. Such variants includeplacing the enzyme, substrate, and sample in the well of a microtiterplate and measuring fluorescence with a microtiter plate reader. Inanother variation, the enzyme is attached to a solid support. When theenzyme is attached to a solid support, it is preferable that a linker isused. An exemplary linking system is biotin/streptavidin. For example,the biotin molecule may be linked to the enzyme and a plate may becoated with streptavidin. When linking an enzyme to a solid support, itis important to determine the effect of linkage on the enzymaticactivity of the enzyme.

[0073] In an alternative embodiment, the solid support may be a bead andfluorescence measured using a flow cytometer. In embodiments having theenzyme attached to a solid support, the biosensor may be reusable. Oldsubstrate and sample is removed, leaving the enzyme in place. Newsubstrate and sample may then be added.

[0074] In another embodiment, the nucleic acid enzyme may be used inconjunction with fiber-optics (Lee & Walt, 2000). The nucleic acidenzyme may be immobilized on the surface of silica microspheres anddistributed in microwells on the distal tip of an imaging fiber. Theimaging fiber may then be coupled to a epifluorescence microscopesystem.

[0075] In certain embodiments, the biosensor will comprise an array ofnucleic acid enzymes. The arrays of the present invention provide forthe simultaneous screening of a variety of ion by nucleic acid enzymes.The array may contain as little as 2 or as many as 10,000 differentnucleic acid enzymes. Of course, any integer in between may be used.Preferably, each individual nucleic acid enzyme has a measurabledifference in specificity or affinity for at least one ion compared toat least one other nucleic acid enzyme within the array.

[0076] In preferred embodiments, the array is a high-density array likethose used in DNA-chip technologies. Methods of forming high densityarrays of nucleic acids with a minimal number of synthetic steps areknown. (U.S. Pat. No. 6,040,138). The nucleic acid array can besynthesized on a solid support by a variety of methods, includinglight-directed chemical coupling, and mechanically directed coupling(U.S. Pat. No. 5,143,854; WO 90/15070; WO 92/10092; WO 93/09668). Usingthis approach, one heterogenous array of polymers is converted, throughsimultaneous coupling at a number of reaction sites, into a differentheterogenous array.

[0077] The light-directed combinatorial synthesis of nucleic acid arrayson a glass surface uses automated phosphoramidite chemistry and chipmasking techniques. In one specific implementation, a glass surface isderivatizied with a silane reagent containing a functional group, e.g.,a hydroxyl or amine group blocked by a photolabile protecting group.Photolysis through a photolithogaphic mask is used selectively to exposefunctional groups which are then ready to react with incoming5′-photoprotected nucleoside phosphoramidites. The phosphoramiditesreact only with those sites which are illuminated (and thus exposed byremoval of the photolabile blocking group). Thus, the phosphoramiditesonly add to those areas selectively exposed from the preceding step.These steps are repeated until the desired array of sequences have beensynthesized on the solid surface. Combinatorial synthesis of differentnucleic acid analogues at different locations on the array is determinedby the pattern of illumination during synthesis and the order ofaddition of coupling reagents.

[0078] In the event that a PNA is used in the procedure, it is generallyinappropriate to use phosphoramidite chemistry to perform the syntheticsteps, since the monomers do not attach to one another via a phosphatelinkage. Instead, peptide synthetic methods are substituted (U.S. Pat.No. 5,143,854).

[0079] In addition to the foregoing, additional methods which can beused to generate an array of nucleic acids on a single solid support areknown (For example, WO 93/09668). In these methods, reagents aredelivered to the solid support by either (1) flowing within a channeldefined on predefined regions or (2) “spotting” on predefined regions.However, other approaches, as well as combinations of spotting andflowing, may be employed. In each instance, certain activated regions ofthe solid support are mechanically separated from other regions when themonomer solutions are delivered to the various reaction sites.

[0080] A typical “flow channel” method applied to the nucleic acidenzyme arrays of the present invention can generally be described asfollows. Diverse nucleic acid sequences are synthesized at selectedregions of a solid support by forming flow channels on a surface of thesolid support through which appropriate reagents flow or in whichappropriate reagents are placed. For example, assume a monomer “A” is tobe bound to the solid support in a first group of selected regions. Ifnecessary, all or part of the surface of the solid support in all or apart of the selected regions is activated for binding by, for example,flowing appropriate reagents through all or some of the channels, or bywashing the entire solid support with appropriate reagents. Afterplacement of a channel block on the surface of the solid support, areagent having the monomer A flows through or is placed in all or someof the channel(s). The channels provide fluid contact to the firstselected regions, thereby binding the monomer A on the solid supportdirectly or indirectly (via a spacer) in the first selected regions.

[0081] Thereafter, a monomer B is coupled to second selected regions,some of which may be included among the first selected regions. Thesecond selected regions will be in fluid contact with a second flowchannel(s) through translation, rotation, or replacement of the channelblock on the surface of the solid support; through opening or closing aselected valve; or through deposition of a layer of chemical orphotoresist. If necessary, a step is performed for activating at leastthe second regions. Thereafter, the monomer B is flowed through orplaced in the second flow channel(s), binding monomer B at the secondselected locations. In this particular example, the resulting sequencesbound to the solid support at this stage of processing will be, forexample, A, B, and AB. The process is repeated to form a vast array ofnucleic acid enzymes of desired length and sequence at known locationson the solid support.

[0082] After the solid support is activated, monomer A can be flowedthrough some of the channels, monomer B can be flowed through otherchannels, a monomer C can be flowed through still other channels, etc.In this manner, many or all of the reaction regions are reacted with amonomer before the channel block must be moved or the solid support mustbe washed and/or reactivated. By making use of many or all of theavailable reaction regions simultaneously, the number of washing andactivation steps can be minimized.

[0083] One of skill in the art will recognize that there are alternativemethods of forming channels or otherwise protecting a portion of thesurface of the solid support. For example, according to someembodiments, a protective coating such as a hydrophilic or hydrophobiccoating (depending upon the nature of the solvent) is utilized overportions of the solid support to be protected, sometimes in combinationwith materials that facilitate wetting by the reactant solution in otherregions. In this manner, the flowing solutions are further preventedfrom passing outside of their designated flow paths.

[0084] The “spotting” methods of preparing nucleic acid arrays can beimplemented in much the same manner as the flow channel methods. Forexample, a monomer A can be delivered to and coupled with a first groupof reaction regions which have been appropriately activated. Thereafter,a monomer B can be delivered to and reacted with a second group ofactivated reaction regions. Unlike the flow channel embodimentsdescribed above, reactants are delivered by directly depositing (ratherthan flowing) relatively small quantities of them in selected regions.In some steps, of course, the entire solid support surface can besprayed or otherwise coated with a solution. In preferred embodiments, adispenser moves from region to region, depositing only as much monomeras necessary at each stop. Typical dispensers include a micropipette todeliver the monomer solution to the solid support and a robotic systemto control the position of the micropipette with respect to the solidsupport. In other embodiments, the dispenser includes a series of tubes,a manifold, an array of pipettes, or the like so that various reagentscan be delivered to the reaction regions simultaneously.

[0085] Methods of detecting fluorescent signals on a DNA chip are wellknown to those of skill in the art. In a preferred embodiment, thenucleic acid enzyme array is excited with a light source at theexcitation wavelength of the particular fluorescent label and theresulting fluorescence at the emission wavelength is detected. In aparticularly preferred embodiment, the excitation light source is alaser appropriate for the excitation of the fluorescent label.

[0086] A confocal microscope may be automated with a computer-controlledstage to automatically scan the entire high density array. Similarly,the microscope may be equipped with a phototransducer (e.g., aphotomultiplier, a solid state array, a ced camera, etc.) attached to anautomated data acquisition system to automatically record thefluorescence signal produced by each nucleic acid enzyme on the array.Such automated systems are described at length in U.S. Pat. No.5,143,854 and PCT application 20 92/10092.

EXAMPLES

[0087] The following examples are included to demonstrate embodiments ofthe invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples that follow representtechniques discovered by the inventors to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments that are disclosed and still obtainlike or similar results without departing from the spirit and scope ofthe invention.

Example 1 In Vitro Selection of a Ion-Dependent Deoxyribozyme

[0088] This example demonstrates a method of creating nucleic acidenzymes that are dependent on the presence of an ion for activity. Morespecifically, use of a partially random DNA library to obtaindeoxyribozymes that cleave RNA in the presence of Zn²⁺ or Co²⁺ is shown.

[0089] Materials and Methods Used in this Example

[0090] Oligonucleotides

[0091] DNA oligonucleotides were purchased from Integrated DNATechnologies Inc. Sequences of the random DNA template and the primers(P1, P2 and P3) used in PCR amplifications are listed below: P1:5′-GTGCCAAGCTTACCG-3′ (SEQ ID NO: 3) P2:5′-CTGCAGAATTCTAATACGACTCACTATAGGAA (SEQ ID NO: 4) GAGATGGCGAC-3′ P3:5′-GGGACGAATTCTAATACGACTCACTATrA-3′ (SEQ ID NO: 5)

[0092] Template for Random DNA Pool: 5′-GTGCCAAGCTTACCGTCAC-N40-GAGATCTC(SEQ ID NO: 6) GCCATCTCTTCCTATAGTGAGTCGTATTAG-3′

[0093] Primer P1b and P3b are the 5′-biotinylated version of primers P1and P3. Primer P1a and P3a were prepared by 5′-labeling P1 and P3 with[γ-³²P] ATP (Amersham) and T4 polynucleotide kinase (Gibco). The DNA/RNAchimeric substrate (17DS) for trans-cleavage assays has the sequence5′-ACTCACTATrAGGAAGAGATG-3′ (SEQ ID NO:2), where rA denotes a singleribonucleotide. The all-RNA substrate (17RS) with the same sequence waspurchased from Dharmacon Research Inc. The trans-cleaving deoxyribozyme17E has the sequence 5′-CATCTCTTCTCCGAGCCGGTCGAAATAGTGAGT-3′ (SEQ IDNO:1). The deoxyribozyme named 17E1 is a variant of 17E with thesequence 5′-CATCTCTTTTGTCAGCGACTCGAAATAGTGA GT-3′ (SEQ ID NO:7). Alloligonucleotides were purified using denaturing polyacrylamide gelelectrophoresis and desalted with the SepPak nucleic acid purificationcartridges (Waters) before use.

[0094] Preparation of Random DNA Pool

[0095] The initial pool for DNA selection was prepared bytemplate-directed extension followed by PCR amplification. The extensionwas carried out with 200 pmol of DNA template containing a 40-nucleotiderandom sequence region, and 400 pmol of primer P3b in 20×100 μl reactionmixtures for four thermal-cycles (1 min at 92° C., 1 min at 52° C., and1 min at 72° C.). Reaction buffer also included 0.05 U/μl Taq polymerase(Gibco), 1.5 mM MgCl₂, 50 mM KCl, 10 mM Tris-HCl (pH 8.3 at 25° C.),0.01% gelatin and 0.2 mM of each dNTP. Subsequently, 1 nmol each of P1and P3b were added to the extension product to allow four more cycles ofPCR amplification. The products were precipitated with ethanol anddissolved in 0.5 ml of buffer A, which contains 50 mM HEPES (pH 7.0),500 mM (for Zn-DNA selection) or 1 M (for Co-DNA selection) NaCl. About20 μM EDTA was also added to the buffer to chelate trace amount ofdivalent metal ion contaminants.

[0096] In Vitro Selection

[0097] The random DNA pool was immobilized on a NeutrAvidin column(Pierce) by incubating with the column materials for 30 minutes. Themixture was gently vortex-mixed a few times during the incubation. Theunbound DNA strands were eluted with at least 5×100 μl of buffer A. Thenon-biotinylated strands of immobilized DNA were washed off the columnwith 5×100 μl of freshly prepared 0.2 M NaOH and 20 μM EDTA. The columnwas then neutralized with 5×100 μl of buffer A The cleavage reaction wascarried out by incubating the immobilized single-stranded DNA containingthe single ribonucleotide (rA) with 3×20 μl of reaction buffer (buffer Aplus 1 mM ZnCl₂ or CoCl₂) over 1 h. The eluted DNA molecules were pooledand precipitated with ethanol. A fraction of the selected DNA wasamplified in 100 μl PCR reaction with 40 pmol each of primers P1 and P2over 10-20 thermal cycles. One tenth of the PCR product was furtheramplified for six cycles with 50 pmol of primers P1 and P3b. The finalPCR product was ethanol precipitated and used to initiate the next roundof selection. During the selection of Zn(II)-dependent deoxyribozymes(called Zn-DNA hereafter), the concentration of ZnCl₂ was kept constantat 100 μM in the reaction buffer for the following rounds of selection.Reaction time was gradually decreased from 1 h to 30 s within 12 roundsof selection. For the selection of Co(II)-dependent deoxyribozymes(called Co-DNA hereafter), the concentration of CoCl₂ was graduallydecreased from 1 mM to 100 μM and the reaction time from 1 h to 1 minwithin 10 rounds of selection. The twelfth generation of selectivelyamplified Zn-DNA and the tenth generation of Co-DNA were cloned usingTA-TOPO Cloning Kit (Invitrogen) and sequenced with T7 Sequenase 2.0Quick-denatured Plasmid Sequencing Kit (Amersham).

[0098] Reselection

[0099] Based on the sequence of class I Zn-DNA or Co-DNA, partiallydegenerate DNA template libraries for reselection were synthesized(Integrated DNA Technology Inc.) with 20% degeneracy at the N40 region.In other words, during the oligonucleotide synthesis of the N40 region,the wild type sequence was introduced at a probability of 80% at eachposition, while the other three nucleotides each occurred at aprobability of 6.67%. The reselection pool was prepared with 10 pmol oftemplate and 100 pmol of primers P1 and P3b using the same protocolpreviously described. With 10 pmol (number of molecules S=6×10¹²) ofpartially randomized template, the statistic parameters of the DNAlibrary used for reselection were calculated based on the followingequations.

P(k,n,d)=[n!/(n−k)!k!]d ^(k)(1−d)^(n−k)  (1)

N(k)=[n!/(n−k)!k!]3^(k)  (2)

C(n,k)=SP(k,n,d)/N(k)  (3)

[0100] P(k,n,d) is the probability of having k mutations within n(number of randomized positions, n=40) nucleotide positions that havebeen randomized at a degeneracy of d. N(k) is the number of distinctsequences that have k mutations with respect to the prototype sequence.C(n,k) is the number of copies for each sequence that has k mutations.The reselection pool was expected to contain the wild type sequence, allpossible sequences with 1-8 point mutations, and a sampling of thesequences with >8 point mutations. More than half of the populationcontains ≧8 point-mutations. The protocol for reselection was the sameas the primary selection, except that the reaction time was decreasedfrom 20 min to 1 min and the concentration of ZnCl₂ or CoCl₂ wasdecreased from 20 μM to 5 μM over six generations. The sixth generationof reselected Zn- or Co-DNA were cloned and sequenced as previouslydescribed.

[0101] Kinetic Assays of the Reselected Cis-Cleaving DNA

[0102] The 5′ ³²P-labeled precursor DNA for cis-cleavage assay wasprepared by PCR-amplification of the selected DNA population or thecloned DNA plasmid with primer 1b and 3a. The double-stranded productwas immobilized on a NeutrAvidin column through the biotin moiety onprimer P1b. The catalytic strand of DNA was eluted off the column with3×20 μl freshly prepared 0.2 N NaOH and neutralized with 8 μl of 3 Msodium acetate (pH 5.3) in the presence of 50 μg/ml bovine serum albumin(Sigma). Following ethanol precipitation, the single-stranded DNA waspurified on an 8% denaturing polyacrylamide gel and desalted with SepPaknucleic acid purification cartridge. Bovine serum albumin (50 μg/ml) wasadded to the gel-soaking buffer (0.2 M NaCl, 20 μM EDTA, 10 mM Tris-HCl,pH 7.5) to prevent the DNA from adhering to the tube. The concentrationof the DNA was determined by scintillation counting the radioactivity.

[0103] The precursor DNA was dissolved in buffer A and incubated at roomtemperature for 10 min before CoCl₂ or ZnCl₂ was added. The reaction wasstopped with 50 mM EDTA, 90% formamide and 0.02% bromophenol blue.Reaction products were separated on an 8% denaturing polyacrylamide geland quantified with a Molecular Dynamic phosphorimager.

[0104] In vitro Selection of Zn(II)- or Co(II)-Dependent Deoxyribozymes

[0105] The DNA molecules capable of cleaving an RNA bond in the presenceof Co²⁺ or Zn²⁺ were obtained through in vitro selection. The initialDNA library for selection contains ˜10¹⁴ out of the possible 10²⁴ (=4⁴⁰)DNA sequences. These molecules consist of a random sequence domain of 40nucleotides flanked by two conserved primer-binding regions. Thesequence of the conserved region was designed in such a way that theycould form two potential substrate-binding regions (FIG. 1A). Aribonucleic adenosine was embedded in the 5′-conserved sequence regionand was intended to be the cleavage site, since an RNA bond is moresusceptible than a DNA bond toward hydrolytic cleavage. The intrinsichalf-life of the phosphodiester linkage in RNA at pH 7 and 25° C. isestimated to be 1,000 years. The corresponding value for DNA is 200million years.

[0106] The DNA pool was immobilized on a NeutrAvidin column through thebiotin moiety on the 5 terminus of the DNA. Biotin and Avidin bindstrongly with an association constant of K_(a)=10¹⁵ M⁻¹. The sequencesthat underwent self-cleavage in the presence of Co²⁺ or Zn²⁺ were elutedoff the column, amplified and used to seed the next round of selection(FIG. 1B). The selection stringency was increased during the selectionprocess with shorter reaction time and less available divalent metalions. The activity of the selected Zn-DNA gradually increased until thetwelfth generation and declined thereafter, while the highest activitywas achieved with the tenth generation of Co-DNA. Therefore the twelfthgeneration of Zn-DNA and the tenth generation of Co-DNA were cloned andsequenced. The cloned sequences can be divided into different classesbased on sequence similarity (FIG. 2 and FIG. 3).

[0107] Individual sequences of the cloned Zn-DNA and Co-DNA wererandomly chosen and sampled for activity. Under the selection conditions(100 μM Zn²⁺, 500 mM NaCl, 50 mM HEPES, pH 7.0, 25° C.), the observedrate constants of Zn-DNAs from sequence-classes I and II were 0.1-0.2min⁻¹, while class III sequences were less active, with k_(obs) around0.02 min⁻¹. The cleavage rate of the initial pool was 2×10⁻⁷ min⁻¹.Therefore, a 10⁵-10⁶ fold increase in cleavage rate has been achieve forZn-DNA selection. The cleavage rates of all the randomly picked Co-DNAsequences were <0.02 min⁻¹ under the conditions for Co-DNA selections(100 μM Co²⁺, 1 M NaCl, 50 mM HEPES, pH 7.0, 25° C.). Interestingly,even in the buffer (1 M NaCl, 50 mM HEPES, pH 7.0) alone, the class IICo-DNA exhibited similar activity as in the presence of 100 μM Co²⁺ orZn²⁺.

[0108] Clone #5 of Zn-DNA (Zn-5) and clone #18 of Co-DNA (Co-18) showedrelatively high activity, as well as high frequency of occurrence,within their lineages. The k_(obs) were 0.17 min⁻¹ for Zn-5 (in 100 μMZn²⁺) and 0.02 min⁻¹ for Co-18 (in 100 μM Co²⁺). The sequences of Zn-5and Co-18 were partially randomized (see Material and Methods fordetails) and subjected to reselection in order to further improve thereactivity and metal-binding affinity, and to explore the sequencerequirement of the conserved catalytic motif Based on equations (1)-(3),the reselection pool was expected to contain the wild type sequence, allpossible sequences with 1-8 point mutations, and a sampling of thesequences with >8 point mutations. More than half of the populationshould contain ≧8 point mutations. Six rounds of reselection werecarried out with 5-20 μM Zn²⁺ or Co²⁺, however the activity of thereselected DNA was similar to the activity of the wild type sequences.Sequencing of the Zn-DNA from both the initial selection and reselectionrevealed a highly conserves sequence region. Therefore the lack ofactivity improvement after reselection likely reflects a sequence pooldominated by a few highly reactive sequences.

[0109] Sequence Alignment and Structure Analysis of Zn-DNA

[0110] The sequences of thirty individual clones of initially selectedZn-DNA can be divided into three major classes based on sequencesimilarity. Differences among members of each class were limited to afew point mutations (FIG. 2). A highly conserved sequence region of 20nt, 5′-TX₁X₂X₃AGCY₁Y₂Y₃TCGAAATAGT-3′ (SEQ ID NO:8) (Region-20nt), wasobserved in all but one sequence albeit at different locations. Thesequences of 5′-X₁X₂X₃-3′ and 3′-Y₃Y₂Y₁-5′ are complimentary andcovariant, indicating that they form base pair with each other:

[0111] 5′-X₁X₂X₃-3′

[0112] 3′-Y₃Y₂Y₁-5′

[0113] The secondary structures of the sequenced Zn-DNA were predictedusing Zuker's DNA mfold program (seehttp://mfold.wustl.edu/˜folder/dna/form1.cgi) through minimization offolding energy. The most stable structures predicted for thosecontaining Region-20nt all contained a similar structure motif Thiscommon motif consists of a pistol-shaped three-way helical junctionformed by a 3 bp hairpin, an 8 bp hairpin and a double helix linking tothe rest of the molecule. The 3 bp hairpin and its adjacentsingle-stranded regions are part of the Region-20nt. The ribonucleicadenosine is unpaired and positioned opposite of the 3 bp hairpin.

[0114] After reselection, twenty-eight Zn-DNA clones were sequenced(FIG. 4). When compared with the parental wild type sequence (class IZn-DNA), the reselected Zn-DNA contained point mutations mostly outsideof Region-20nt. About one third of these sequences have a T→A mutationat position 73, converting the T-T mismatch in the wild type sequence toa Watson-Crick base pair. In one fourth of the reselected DNAs, the 5nucleotide single-stranded bulge of the three-way junction has thesequence 5′-ACGAA-3′, corresponding to 5′-TCGAA-3′ in the wild type.Clone #17 (named ZnR17) of the reselected Zn-DNA is most active underselection conditions (FIG. 4). Structural analysis of ZnR17 revealed twocompleted base-paired helices in the three-way junction. Therefore, itwas engineered into a trans-cleaving deoxyribozyme by deleting thesequences outside of the three-way junction and the loop of the 8 bphairpin. Such truncation resulted in two individual stands, whichhybridize with each other through two 9-10 bp helices. The strandcontaining the single ribonucleotide residue (rA) is considered as thesubstrate (named 17DS), while the other strand as the enzyme (named17E). The catalytic core, which was highly conserved during selection,consists of a 3 bp hairpin and a 5 nt single-stranded bulge (FIG. 5).

[0115] Although ZnR17 was selected in Zn²⁺, it does not containstructure motifs that were discovered in several Zn(II)-binding RNAmolecules (Ciesiolka et al., 1995; Ciesiolka & Yarus, 1996). However,the conserved region of ZnR17 is very similar to that of the 8-17deoxyribozymes selected by Santoro and Joyce using Mg²⁺ as cofactor(Santoro & Joyce, 1997). The unpaired bulge region in the 8-17 DNAenzyme has the sequence 5′-WCGR-3′ or 5′-WCGAA-3′ (W=A or T; R=A or G).The highest activity was observed with the sequence containing5′-TCGAA-3′. Among the Zn(II)-dependent deoxyribozymes we obtained afterreselection, 85% of them fell within the 5′-WCGAA-3′ regime (W=A or T).However, the sequence of the two double helices flanking the catalyticcore is different between the 8-17 (FIG. 6D) and the 17E deoxyribozymes(FIG. 6F), reflecting their different designs of the selection pool.Similar sequence motif was also observed in an RNA-cleavingdeoxyribozyme (named Mg5) selected by Faulhammer and Famulok using 50 mMhistidine and 0.5 mM Mg²⁺ as cofactors (Faulhammer & Famulok, 1997). Thehomologous region in 31 out of the 44 sequenced clones had the sequence5′-TX₁X₂X₃AGCY₁Y₂Y₃ACGAA-3′ (SEQ ID NO:9), falling within the WCGAA-3′regime. The authors predicted a secondary structure different from the8-17 or 17E motif based on chemical modification analysis. However, astructure containing a three-way junction similar to that of the 17E and8-17 deoxyribozymes is consistent with the chemical mapping results.

[0116] Sequence Alignment and Structure Analysis of Co-DNA

[0117] The sequences of the cis-cleaving deoxyribozyme selected in thepresence of Co²⁺ are more diverse than the Zn-DNA. They can be dividedinto three major classes based on sequence similarity (FIG. 3). There isno consensus sequence region among different classes. The secondarystructure of each sequence class of Co-DNA was predicted with DNA mfoldprogram. The minimal conserved sequence motif of class I Co-DNA includesa bulged duplex. The cleavage site is within the 13 nt single-strandedbulge. A 4 bp hairpin is also highly conserved and linked to the bulgedduplex through 3 unpaired nucleotides. The folding of the sequencesoutside of this minimal motif was highly variable and resulted instructures with a wide range of stabilization energy.

[0118] The class II Co-DNA contains a sequence region(5′-ACCCAAGAAGGGGTG-3′ (SEQ ID NO:10)) that was also found in anRNA-cleaving deoxyribozyme (termed G3) selected by Geyer and Sen (1997)(FIGS. 7A and 7B). The minimal motif predicted for class II Co-DNA showssimilarity to that proposed for the G3 deoxyribozyme as well. The G3deoxyribozyme was believed to be fully active in the absence of anydivalent metal ions. Copious use of divalent metal chelating agents,such as EDTA, and accurate trace-metal analysis of the reactionsolutions were used to rule out the need of the G3 deoxyribozyme forcontaminating levels of divalent metals. As mentioned earlier, theactivity of class II Co-DNA was the same in buffer alone or with addedCo²⁺ or Zn²⁺, suggesting that this class of Co-DNA most likely containthe divalent metal-independent structure motif

[0119] Effect of Metal Ions on the Activity of the Cis-CleavingDeoxyribozymes

[0120] ZnR17 and Co-118 were examined for their activity dependence onmonovalent ions and divalent metal ions other than Zn²⁺ and Co²⁺. In thepresence of 1 mM EDTA and without added Zn²⁺ ions, no cleavage wasobserved with ZnR17 even after two days, strongly suggesting thatdivalent metal ions are required for the activity of ZnR17. Although thecis-cleaving Zn-DNA was selected in the presence of 500 mM NaCl, NaClwas actually inhibitory to enzymatic activity. With 0-2 M NaCl added tothe reaction buffer (100 μM Zn²⁺, 50 mM HEPES, pH 7.0), k_(obs)decreased with increasing NaCl concentration. The deleterious effect ofNaCl was also manifested by lowered final percentage of cleavageproducts. For instance, only 50% of ZnR17 could be cleaved in thepresence of 2 M NaCl even after long incubation times, while >95% of theDNA was cleavable in the absence of extra NaCl. Contrary to the Zn-DNA,the activity of Co-18 relies on NaCl and no cleavage was observed in theabsence of NaCl. With 1 M NaCl, 8% of Co-18 molecules were cleavedwithin 5 min, while <0.2% were cleaved in the absence of extra NaCl.

[0121] Although the deoxyribozymes were selected using either zinc orcobalt as cofactor, they are also active in other transition metal ionsand in Pb²⁺. The cleavage efficiency of ZnR17 followed the trend ofPb²⁺>Zn²⁺>Mn ²⁺˜Co²⁺˜Ca²⁺>Cd²⁺>>Ni²⁺>Mg²⁺. It is noteworthy that thecleavage rate in Ca²⁺ was much higher than in Mg²⁺, a similar trend wasobserved with the Mg5 deoxyribozyme. The order of Co-18 activity is asfollow: Zn²⁺>Pb²⁺˜Co²⁺>Ni²⁺>Cd²⁺˜Mn²⁺>Mg²⁺˜Ca²⁺. In general, both ZnR17and Co-18 are more active in transition metal ions than inalkaline-earth metals, and higher activities were achieved with Pb²⁺,Co²⁺ and Zn²⁺. The preference of the selected deoxyribozymes for Co²⁺and Zn²⁺ reflected their selection criteria. A similar trend(Pb²⁺>Zn²⁺>Mn²⁺>Mg²⁺) was also observed with all four RNA-cleavingdeoxyribozymes selected in parallel by Breaker and Joyce using one ofthe four metal ions (Pb²⁺, Zn²⁺, Mn²⁺, Mg²⁺) as cofactor (1995). Theproposed secondary structures of the deoxyribozymes selected in Pb²⁺ andMg²⁺ have been reported (Breaker & Joyce, 1994, 1995). No structuresimilarity was observed between ZnR17 and those RNA-cleavingdeoxyribozymes.

SUMMARY

[0122] Using in vitro selection technique, several groups ofRNA-cleaving deoxyribozymes were isolated using Zn²⁺ or Co²⁺ ascofactor. No common sequence or structural features were observedbetween the Co(II)-dependent and the Zn(II)-dependent deoxyribozymes, inspite of the chemical similarities between these two transition metalions. The deoxyribozymes selected in Zn²⁺ share a common motif with the8-17 and the Mg5 deoxyribozymes isolated under different conditions,including the use of different cofactors. Both the Co-DNA and the Zn-DNAexhibited higher activity in the presence of transition metal ions thanin alkaline earth metal ions, which are the most commonly adopted metalcofactors by naturally occurring ribozymes.

Example 2 Deoxyribozyme as a Biosensor for Pb²⁺ Detection

[0123] This Example describes a fluorescence-based biosensor for thedetection of Pb²⁺. The biosensor utilizes a deoxyribozyme developed inExample 1 (termed 17E) combined with fluorescence technology to allowquantitative and real time measurements of catalytic activity. Becausecatalytic activity is dependent on Pb²⁺, the biosensor providesreal-time, quantitative, and sensitive measurements of Pb²⁺concentrations.

[0124] Materials and Methods used in this Example

[0125] Oligonucleotides

[0126] The oligonucleotides were purchased from Integrated DNATechnology Inc. The cleavable substrate (Rh-17DS) is a DNA/RNA chimerawith the sequence 5′-ACTCACTATrAGGAAGAGATG-3′ (SEQ ID NO:2), in which rArepresents a ribonucleotide adenosine. This RNA base is replaced with aDNA base for the non-cleavable substrate (Rh-17DDS) (SEQ ID NO:11) usedin the control experiment. Both substrates are covalently linked at the5′ end with 6-carboxytetramethylrhodamin through NHS-ester conjugation.The deoxyribozyme (17E-Dy) is labeled at the 3′-end with Dabcyl via CPGphosphoramidite and has the sequence5′-CATCTCTTCTCCGAGCCGGTCGAAATAGTGAGT-3′ (SEQ ID NO:1). All theoligonucleotides were purified by denaturing 20% polyacrylamide gelelectrophoresis to ensure 100% labeling with the fluorescent dyes.

[0127] Fluorescence Spectroscopy

[0128] The enzyme-substrate complex was prepared with 50 nM each of17E-Dy and Rh-17DS in 50 mM NaCl, 50 mM HEPES (pH 7.5) with a volume of600 μl. The sample was heated at 90° C. for 2 min and cooled to 5° C.over 15 min to anneal the enzyme and substrate strands together.Steady-state and slow kinetic fluorescence spectra were collected with aSLM 8000S photon counting fluorometer. Polarization artifacts wereavoided by using “magic angle” conditions. The steady-state emissionspectra were collected from 570 to 700 nm (λ_(ex)=560 nm). Thetime-dependent DNA enzyme catalyzed substrate cleavage was monitored at580 nm at 2 s intervals. To initiate the reaction, 1-2 μl ofconcentrated divalent metal ion solution was injected into the cuvetteusing a 10 μl syringe while the DNA sample in the cuvette was constantlystirred.

[0129] DNA-Based Sensor of Metal Ions

[0130] An in vitro selected DNA enzyme from Example 1 (termed 17E) thatis capable of cleaving a lone RNA linkage within a DNA substrate (termed17DS) (FIG. 5) was chosen for use as a DNA-based, fluorescent biosensorof metal ions. Assays of this enzyme indicate a highly Pb²⁺ dependentactivity with k_(obs)=6.5 min⁻¹ at pH 6.0 and K_(apparent)=13.5 μM³⁵.The fluorosensor was constructed by labeling the 5′-end of the substratewith the fluorophore 6-carboxytetramethylrhodamin (TAMRA) and the 3′-endof the enzyme strand with 4-(4′-dimethylaminophenylazo)benzoic acid)(Dabcyl). Dabcyl serves as a universal fluorescence quencher.Steady-state fluorescence spectra were obtained by exciting the sampleat 560 nm and scanning its emission from 570 to 700 nm.

[0131] When the substrate (Rh-17DS) was hybridized to the enzyme strand(17E-Dy), the fluorescence of TAMRA was quenched by the nearby Dabcyl(FIG. 8). Upon addition of Pb²⁺, this quenching was eliminated and thefluorescence of TAMRA increased by ˜400%. Little change in thefluorescence signal occurred with addition of Pb²⁺ to the substratealone or to the complex of the enzyme and a non-cleavable all DNAsubstrate with identical sequence. These findings show that the changein fluorescent signal with Rh-17DS/17E-Dy results from a DNAenzyme-catalyzed substrate cleavage, followed by product release.

[0132] The substrate cleavage reaction was monitored in real time withfluorescence spectroscopy. Like the ratiometric, anisotropy, orlifetime-based method, kinetic fluorescence measurement is independentof sampling conditions such as illumination intensity and samplethickness (Oehme & Wolfbeis, 1997). In order to determine theselectivity of the catalytic DNA sensor, the fluorescence change (λ=580nm, λ_(ex)=560 nm) of Rh-17DS/17E-Dy upon addition of nine differentdivalent metal ions that are known to be active toward DNA/RNA cleavage(FIG. 9A) was monitored. At the same concentration, Pb²⁺ caused the mostrapid change in fluorescence with a rate of 380 counts·s⁻¹ at 500 nMPb²⁺, pH 7.5. The sensitivity toward Pb²⁺ was >80 times higher thanother divalent metal ions (FIG. 9A). Remarkably, this trend ofselectivity was maintained even under simulated physiological conditionscontaining 100 mM NaCl, 1 mM Mg²⁺, and 1 mM Ca²⁺ (FIG. 9B). Furthermore,the signal response to Pb²⁺ was not affected by the presence of equalamounts of each of these divalent metal ions. Therefore, this DNA enzymesensor is well suited for selective monitoring Pb²⁺ in the presence ofother metal ions.

[0133] In addition to the selectivity of the DNA enzyme probe for Pb²⁺over other metal ions, the range of Pb²⁺ concentrations which give riseto a fluorescent response is also important. As shown in FIG. 10A, therate of fluorescence change increased with Pb²⁺ concentration up to 4μM. The detection limit for Pb²⁺ is around 10 nM, 50 fold less than thetoxic level defined by the Center for Disease Control.

Example 3 DNA Chip Comprising an Array of Nucleic Acid Enzymes

[0134] This prophetic example describes the production of and use of aDNA chip for sensing ions, in particular heavy metal ions.

[0135] The first step towards the application of deoxyribozymes in heavymetal sensing is to obtain various deoxyribozymes with different metalspecificity and affinity. In vitro selection will be carried out toisolate a variety of deoxyribozymes. A detailed description of theselection protocol can be found in Example 1. Each family ofdeoxyribozyme will be specific for different divalent metal ions (e.g.Pb²⁺, Hg²⁺, Zn²⁺, Co²⁺, Cd²⁺, Ni²⁺, Mn²⁺, etc). Within each family,different sequences will have different affinities of the specifiedmetal ion.

[0136] These deoxyribozymes and their substrates will then be arrayedonto a DNA chip with one dimension for metal ion specificity and theother for affinity of the corresponding metal (FIG. 11). The enzymestrands immobilized on the chip at 3′-ends can be synthesized on thechip using photolithographic methods (Fodor et al., 1991; Pease et al.,1994) or can be synthesized off-chip and then attached to the chip usingvarious methods (Joos et al., 1997; O'Donnell-Maloney et al., 1997;Guschin et al., 1997). The 5′-end of the enzyme strand will be labeledwith a fluorophore. The 3′-end of the substrate strand will be labeledwith a fluorescence quencher, which can be a fluorescent ornon-fluorescent moiety. For example, guanidine base is an efficientquencher of fluorescein.

[0137] Hybridization of the enzyme and substrate will result in thequenching of the donor fluorescence. Upon exposure to the samplecontaining the active metal ion, the substrate will be cleaved andproducts will dissociate, resulting in strong fluorescence of the dyeattached to the enzyme strand. The metal ion species can bequalitatively identified based on the metal specificity of differentfamilies of deoxyribozymes. A hypothetical sample result is shown inFIG. 11B. The pattern of fluorescence intensity shows that there arethree kinds of metal (M1, M4, and M6) in the sample.

[0138] The concentration of the metal ion under inspection can bequantified with deoxyribozymes with different metal affinity. Given acertain concentration of the metal ion, different sequences within thesame family will have different cleavage efficiencies due to theirdifferent threshold in response to the metal concentration. The metalconcentration applied may exceed the saturation concentration of thosehaving higher affinity; therefore full cleavage will occur within acertain time and present strong fluorescence. On the other hand, thesubstrates of those with lower affinity will only be partly cleaved andemit weaker fluorescence. The sample hypothetical result shown in FIG.11B shows high (c1), medium (c4), and low (c6) concentrations of M1, M4,and M6, respectively.

[0139] The fluorescence patterns with respect to different deoxyribozymesequences will be compared with standard calibration maps. Afterde-convolution of the fluorescence pattern, direct information can beobtained about the identity and concentration of metal ions in thesamples.

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1 88 1 33 DNA Artificial Sequence Description of Artificial SequenceTrans-cleaving deoxyribozyme 17E 1 catctcttct ccgagccggt cgaaatagtg agt33 2 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic chimeric substrate 2 actcactata ggaagagatg 20 3 15 DNAArtificial Sequence Description of Artificial Sequence Primer 3gtgccaagct taccg 15 4 43 DNA Artificial Sequence Description ofArtificial Sequence Primer 4 ctgcagaatt ctaatacgac tcactatagg aagagatggcgac 43 5 28 DNA Artificial Sequence Description of Artificial SequencePrimer 5 gggacgaatt ctaatacgac tcactata 28 6 97 DNA Artificial SequenceDescription of Artificial Sequence Synthetic DNA Template 6 gtgccaagcttaccgtcacn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnng 60 agatctcgccatctcttcct atagtgagtc gtattag 97 7 33 DNA Artificial SequenceDescription of Artificial Sequence Variant of deoxyribozyme named 17E1 7catctctttt gtcagcgact cgaaatagtg agt 33 8 20 DNA Artificial SequenceDescription of Artificial Sequence Synthetic Zn-DNA 8 tnnnagcnnntcgaaatagt 20 9 15 DNA Artificial Sequence Description of ArtificialSequence Synthetic Zn-DNA sequence 9 tnnnagcnnn acgaa 15 10 15 DNAArtificial Sequence Description of Artificial Sequence Class II Co-DNAsequence 10 acccaagaag gggtg 15 11 20 DNA Artificial SequenceDescription of Artificial Sequence Synthetic Rh-17DDS 11 actcactataggaagagatg 20 12 97 DNA Artificial Sequence Description of ArtificialSequence Synthetic chimeric substrate 12 ctaatacgac tcactataggaagagatggc gacatctcnn nnnnnnnnnn nnnnnnnnnn 60 nnnnnnnnnn nnnnnnnngtgacggtaagc ttggcac 97 13 43 DNA Artificial Sequence Description ofArtificial Sequence Synthetic Zn-DNA 13 ctgcagaatt ctaatacgac tcactataggaagagatggc gac 43 14 50 DNA Artificial Sequence Description ofArtificial Sequence Synthetic Zn-DNA 14 atctcttttg tcagcgactc gaaatagtgtgttgaagcag ctctagtgac 50 15 49 DNA Artificial Sequence Description ofArtificial Sequence Synthetic Zn-DNA 15 agccatagtt ctaccagcgg ttcgaaatagtgaagtgttc gtgactatc 49 16 49 DNA Artificial Sequence Description ofArtificial Sequence Synthetic Zn-DNA 16 ggccatagtt ctaccagcgg ttcgaaatagtgaaatgttc gtgactatc 49 17 51 DNA Artificial Sequence Description ofArtificial Sequence Synthetic Zn-DNA 17 gccagattag ttctaccagc ggttcgaaatagtgaaatgt tcgtgactat c 51 18 50 DNA Artificial Sequence Description ofArtificial Sequence Synthetic Zn-DNA 18 atctccaaag atgccagcat gctattctccgagccggtcg aaatagtgac 50 19 50 DNA Artificial Sequence Description ofArtificial Sequence Synthetic Zn-DNA 19 atctccaaag atgcctgcat gctattctccgagccggtcg aaatagtgac 50 20 50 DNA Artificial Sequence Description ofArtificial Sequence Synthetic Zn-DNA 20 atctcgtctc cgagccggtc gaaatagtcaggtgtttcta ttcgggtgac 50 21 50 DNA Artificial Sequence Description ofArtificial Sequence Synthetic Zn-DNA 21 atcaccttct ccgagccggt cgaaatagtagtttttagta tatctgtgac 50 22 50 DNA Artificial Sequence Description ofArtificial Sequence Synthetic Zn-DNA 22 atctcaggtg ttggctgctc tcgcggtggcgagaggtagg gtgatgtgac 50 23 14 DNA Artificial Sequence Description ofArtificial Sequence Synthetic Zn-DNA 23 ggtaagcttg gcac 14 24 43 DNAArtificial Sequence Description of Artificial Sequence Synthetic Co-DNA24 ctgcagaatt ctaatacgac gcactatagg aagagatggc gac 43 25 50 DNAArtificial Sequence Description of Artificial Sequence Synthetic Co-DNA25 atctcttgta ttagctacac tgttagtgga tcgggtctaa tctcggtgac 50 26 50 DNAArtificial Sequence Description of Artificial Sequence Synthetic Co-DNA26 gtctcttgta ttagctacac tgttagtgga tcgggtctaa tctcggtgac 50 27 50 DNAArtificial Sequence Description of Artificial Sequence Synthetic Co-DNA27 atctcctgta ttagctacac tgttagtgga tcgggtctaa tctcggtgac 50 28 49 DNAArtificial Sequence Description of Artificial Sequence Synthetic Co-DNA28 atctcttgta ttagctacac tgttagtggg aacgttatca ttcggtgac 49 29 45 DNAArtificial Sequence Description of Artificial Sequence Synthetic Co-DNA29 atctcttgac ccaagaaggg gtgtcaatct aatccgtcaa ccatg 45 30 45 DNAArtificial Sequence Description of Artificial Sequence Synthetic Co-DNA30 atctcttgac ccaagaaggg gtgtcaatca aatccgtcaa ccatg 45 31 54 DNAArtificial Sequence Description of Artificial Sequence Synthetic Co-DNA31 atctcttgac ccaagaaggg gtgtcaatct aatccgtaca accatgacgg taag 54 32 52DNA Artificial Sequence Description of Artificial Sequence SyntheticCo-DNA 32 atctcttgac ccaagaaggg gtgtcaatct aatccgtcaa ggatgcggta ag 5233 50 DNA Artificial Sequence Description of Artificial SequenceSynthetic Co-DNA 33 atctcaggtg ttggctgctc ccgcggtggc gggaggtagggtgatgtgac 50 34 50 DNA Artificial Sequence Description of ArtificialSequence Synthetic Co-DNA 34 atctcaggtg ttggcatctc ccgcggtggc gagaggtagggtgatgtgac 50 35 50 DNA Artificial Sequence Description of ArtificialSequence Synthetic Co-DNA 35 atctcaggtg ttggctgctc tcgcggtggc gagaggtagggtcatgtgac 50 36 50 DNA Artificial Sequence Description of ArtificialSequence Synthetic Co-DNA 36 atctcgcagt cgaagcttca ctgttagtgc ggacgggtagacttcgtgac 50 37 50 DNA Artificial Sequence Description of ArtificialSequence Synthetic Co-DNA 37 atttcttctg aatcctcaat gttagtggac ctagtcgtagtcgatgtgac 50 38 50 DNA Artificial Sequence Description of ArtificialSequence Synthetic Co-DNA 38 atctcggagc cagttagcat aatcttctga atcctcaatgttagtgtgac 50 39 50 DNA Artificial Sequence Description of ArtificialSequence Synthetic Co-DNA 39 atctcggtgt tggctggata gagccggtag gccctatcgtagggtgtgac 50 40 50 DNA Artificial Sequence Description of ArtificialSequence Synthetic Co-DNA 40 gtctcttttg tccgcgactc gaaatagtgt gttgaagcagctctagtgac 50 41 54 DNA Artificial Sequence Description of ArtificialSequence Synthetic Co-DNA 41 agccatagtt ctaccagcgg ttcgaaatag tgaagtgttcgtgactatcg gtaa 54 42 14 DNA Artificial Sequence Description ofArtificial Sequence Synthetic Co-DNA 42 ggtaagcttg gcac 14 43 40 DNAArtificial Sequence Description of Artificial Sequence Synthetic Zn-DNA43 ttttgtcagc gactcgaaat agtgtgttga agcagctcta 40 44 40 DNA ArtificialSequence Description of Artificial Sequence Synthetic Zn-DNA 44ttttgtcagc gactcgaaat agtgtgttga agccgctcta 40 45 40 DNA ArtificialSequence Description of Artificial Sequence Synthetic Zn-DNA 45ttttgtcagc gactcgaaat agtgtattgc agtagatcta 40 46 40 DNA ArtificialSequence Description of Artificial Sequence Synthetic Zn-DNA 46ttttgtcagc gactcgaaat agtgtgttac agttgcccta 40 47 40 DNA ArtificialSequence Description of Artificial Sequence Synthetic Zn-DNA 47ttttgtcagc gactcgaaat agagagtcga cacacctctc 40 48 40 DNA ArtificialSequence Description of Artificial Sequence Synthetic Zn-DNA 48ttttgtcagc gactcgaaat agttagttga accagctctc 40 49 40 DNA ArtificialSequence Description of Artificial Sequence Synthetic Zn-DNA 49ttttgtcagc gactcgaaat agtgagtaag aggagctatc 40 50 40 DNA ArtificialSequence Description of Artificial Sequence Synthetic Zn-DNA 50ttttgtcagc gactcgaaat agtgagggga aacagctctc 40 51 39 DNA ArtificialSequence Description of Artificial Sequence Synthetic Zn-DNA 51ttttgtcagc gactcgaaat agttagttga acacctctc 39 52 40 DNA ArtificialSequence Description of Artificial Sequence Synthetic Zn-DNA 52ttttgtcagc gactcgaaat attgagttga agcagatctc 40 53 40 DNA ArtificialSequence Description of Artificial Sequence Synthetic Zn-DNA 53ttttgtcagc gacacgaaat agtgagttga ggcggcgctg 40 54 40 DNA ArtificialSequence Description of Artificial Sequence Synthetic Zn-DNA 54tttttgcagc gacacgaaat agttagttga agaagctctt 40 55 40 DNA ArtificialSequence Description of Artificial Sequence Synthetic Zn-DNA 55ttttgtcagc gactcgaaat agtcagttgt agcagctctt 40 56 40 DNA ArtificialSequence Description of Artificial Sequence Synthetic Zn-DNA 56ttttgtcagc gactcgaaat agtgcgtaga accagctctc 40 57 40 DNA ArtificialSequence Description of Artificial Sequence Synthetic Zn-DNA 57ttttgtcagc gacacgaaat agtgcggtgt atctgccctc 40 58 40 DNA ArtificialSequence Description of Artificial Sequence Synthetic Zn-DNA 58ttttgtcagc gacacgaaat agtgtgatgt agtagctctc 40 59 38 DNA ArtificialSequence Description of Artificial Sequence Synthetic Zn-DNA 59ttttgtcagc gacacgaaat agtgtgacga atcatctc 38 60 39 DNA ArtificialSequence Description of Artificial Sequence Synthetic Zn-DNA 60ttttgtcagc gacacgaaat agtgtgttta agcgctctc 39 61 40 DNA ArtificialSequence Description of Artificial Sequence Synthetic Zn-DNA 61ttttgtcagc gacacgaaat agtgtgttga agcacgtctc 40 62 40 DNA ArtificialSequence Description of Artificial Sequence Synthetic Zn-DNA 62ttttgtcagc gactcgaaat agtttgttga agcagctctc 40 63 40 DNA ArtificialSequence Description of Artificial Sequence Synthetic Zn-DNA 63ttttgtcagc gactcgaaat agtgtattac agcagctctc 40 64 40 DNA ArtificialSequence Description of Artificial Sequence Synthetic Zn-DNA 64ttttgtcagc gactcgaaat agtgtgttga aacagctatc 40 65 40 DNA ArtificialSequence Description of Artificial Sequence Synthetic Zn-DNA 65ttgtgcatgc tactcgtaat tgtgtctcga agcagctctc 40 66 40 DNA ArtificialSequence Description of Artificial Sequence Synthetic Zn-DNA 66gtcagtcagg tactcgaaaa atagtgttca agccgctgtc 40 67 40 DNA ArtificialSequence Description of Artificial Sequence Synthetic Zn-DNA 67tttttgcagc gactcgaaag attgtgttga ggcggctatc 40 68 40 DNA ArtificialSequence Description of Artificial Sequence Synthetic Zn-DNA 68ttctctcagc gactaaaaat agtgtgttga agcccctctc 40 69 40 DNA ArtificialSequence Description of Artificial Sequence Synthetic Zn-DNA 69tattgtcagt gacccaaaat agtatgttga agcagctctg 40 70 39 DNA ArtificialSequence Description of Artificial Sequence Synthetic Zn-DNA 70ttttgtcagc tactgaaata gtgttttgaa gaagtcctg 39 71 15 DNA ArtificialSequence Description of Artificial Sequence Synthetic chimeric substrate71 tcactatagg aagag 15 72 36 DNA Artificial Sequence Description ofArtificial Sequence Synthetic chimeric substrate 72 ctcttcagcgatccggaacg gcacccatgt tagtga 36 73 19 DNA Artificial SequenceDescription of Artificial Sequence Synthetic chimeric substrate 73tcactataag aagagatgg 19 74 37 DNA Artificial Sequence Description ofArtificial Sequence Synthetic chimeric substrate 74 acacatctctgaagtagcgc cgccgtatag tgacgct 37 75 17 DNA Artificial SequenceDescription of Artificial Sequence Synthetic chimeric substrate 75ggagagagau gggugcg 17 76 31 DNA Artificial Sequence Description ofArtificial Sequence Synthetic chimeric substrate 76 cgcacccaggctagctacaa cgactctctc c 31 77 17 DNA Artificial Sequence Description ofArtificial Sequence Synthetic chimeric substrate 77 aaguaacuag agaugga17 78 29 DNA Artificial Sequence Description of Artificial SequenceSynthetic chimeric substrate 78 cgcaccctcc gagccggacg aagttactt 29 79 19DNA Artificial Sequence Description of Artificial Sequence Syntheticchimeric substrate 79 ctcactatag gaagagatg 19 80 41 DNA ArtificialSequence Description of Artificial Sequence Synthetic chimeric substrate80 catctcttaa cggggctgtg cggctaggaa gtaatagtga g 41 81 20 DNA ArtificialSequence Description of Artificial Sequence Synthetic chimeric substrate81 actcactata ggaagagatg 20 82 33 DNA Artificial Sequence Description ofArtificial Sequence Synthetic chimeric substrate 82 catctcttctccgagccggt cgaaatagtg agt 33 83 107 DNA Artificial Sequence Descriptionof Combined DNA/RNA Molecule Synthetic chimeric oligonucleotide 83gggacgaatt ctaatacgac tcactatagg aagagatggc gacaactctt tacccaagaa 60ggggtgngnn nnnngctacn nnatnnnnnt gacggtagct tggcacc 107 84 45 DNAArtificial Sequence Description of Artificial Sequence Synthetic Co-DNA84 cactatagga agagatggcg acatctcttg acccaagaag gggtg 45 85 44 DNAArtificial Sequence Description of Combined DNA/RNA Molecule Syntheticchimeric substrate 85 tgtcaactcg tgactcacta taggaagaga tgtgtcaact cgtg44 86 12 DNA Artificial Sequence Description of Artificial SequencePrimer 86 cacgagttga ca 12 87 33 DNA Artificial Sequence Description ofArtificial Sequence Synthetic oligonucleotide 17E-C 87 catctcttccccgagccggt cgaaatagtg agt 33 88 12 DNA Artificial Sequence Descriptionof Artificial Sequence Primer 88 cacgagttga ca 12

1. A method of detecting the presence of an ion comprising: (a)contacting a nucleic acid enzyme, the enzyme dependent on the ion toproduce a product from a substrate, with a sample suspected ofcontaining an ion; and (b) measuring the product of the nucleic acidenzymatic reaction.
 2. The method of claim 1, wherein the nucleic acidenzyme comprises a ribozyme.
 3. The method of claim 1, wherein thenucleic acid enzyme comprises a deoxyribozyme.
 4. The method of claim 1,wherein the nucleic acid enzyme and the substrate comprise separatenucleic acid strands.
 5. The method of claim 4, wherein the substratecomprises a fluorophore and the enzyme comprises a quencher of thefluorophore.
 6. The method of claim 5, wherein a 5′-end of the substratecomprises the fluorophore.
 7. The method of claim 6, wherein a 3′-end ofthe enzyme comprises the quencher for the fluorophore.
 8. The method ofclaim 5, wherein the fluorophore is TAMRA.
 9. The method of claim 8,wherein the quencher is DABCYL.
 10. The method of claim 5, wherein theenzyme is linked to a support.
 11. The method of claim 4, wherein thesubstrate comprises at least one ribonucleotide.
 12. The method of claim4, wherein the substrate comprises the nucleic acid sequence of SEQ IDNO:2.
 13. The method of claim 4, wherein the enzyme comprises thenucleic acid sequence of SEQ ID NO:1.
 14. The method of claim 3, whereinthe deoxyribozyme comprises a single strand.
 15. The method of claim 14,wherein the single strand comprises a fluorophore.
 16. The method ofclaim 15, wherein the single strand further comprises a quencher for thefluorophore.
 17. The method of claim 14, wherein the single strandcomprises the nucleic acid sequence of SEQ ID NO:1.
 18. The method ofclaim 17, wherein the single strand further comprises the nucleic acidsequence of SEQ ID NO:
 2. 19. The method of claim 14, wherein the singlestrand comprises at least one ribonucleotide.
 20. The method of claim 1,wherein the ion is selected from the group consisting of monovalentions, divalent ions, trivalent ions, and polyvalent ions.
 21. The methodof claim 20, wherein the ion is an anion.
 22. The method of claim 20,wherein the ion is a cation.
 23. The method of claim 22, wherein thecation is a monovalent cation.
 24. The method of claim 23, wherein themonovalent cation is selected from the group consisting of K⁺, Na⁺, Li⁺,Tl⁺, N⁺, and Ag⁺.
 25. The method of claim 22, wherein the cation is adivalent cation.
 26. The method of claim 25, wherein the divalent cationis selected from the group consisting of Mg²⁺, Ca²⁺, Mn²⁺, Co²⁺, Ni²⁺,Zn²⁺, Cd²⁺, Cu²⁺, Pb²⁺, Hg²⁺, Pt²⁺, Ra²⁺, Ba²⁺, and Sr²⁺.
 27. The methodof claim 26, wherein the metal ion is Pb²⁺.
 28. The method of claim 22,wherein the cation is a trivalent cation.
 29. The method of claim 28,wherein the trivalent cation is selected from the group consisting ofCo³⁺, Cr³⁺, and Ln³⁺.
 30. The method of claim 22, wherein the cation isa polyvalent cation.
 31. The method of claim 30, wherein the polyvalentcation is selected from the group consisting of Ce⁴⁺, spermine, andspermidine.
 32. The method of claim 1, wherein the product comprises anucleic acid.
 33. The method of claim 32, wherein the nucleic acidcomprises a fluorophore.
 34. The method of claim 32, wherein the nucleicacid comprises a fluorophore quencher.
 35. The method of claim 1,wherein the sample suspected of containing the ion comprises a watersample.
 36. The method of claim 1, wherein the sample suspected ofcontaining the ion comprises a bodily fluid.
 37. The method of claim 36,wherein the bodily fluid is blood.
 38. The method of claim 1, whereinthe measuring comprises a measurement of fluorescence.
 39. The method ofclaim 38, wherein the measurement of fluorescence is selected from thegroup consisting of fluorescence intensity, fluorescence lifetime, andan isotropy.
 40. The method of claim 39, wherein an increase influorescence is indicative of the presence of the ion.
 41. The method ofclaim 1, wherein an array of nucleic acid enzymes comprises the nucleicacid enzyme.
 42. A method of determining the concentration of an ion ina sample comprising: (a) contacting a nucleic acid enzyme, the enzymedependent on the ion to produce a product from a substrate, with asample containing an unknown concentration of an ion; (b) measuring theproduct of the nucleic acid enzymatic reaction; and (c) comparing themeasurement obtained in (b) with that of a standard curve created usingknown concentrations of the ion.
 43. A biosensor comprising: (a) anucleic acid enzyme dependent on an ion to produce a product; (b) aquencher; and (c) a photodetector.
 44. The biosensor of claim 43comprising an array of nucleic acid enzymes.
 45. The biosensor of claim44, wherein the array comprises nucleic acid enzymes together having arange of ion specificities.
 46. The biosensor of claim 43 furthercomprising a fluorophore.
 47. A biosensor comprising: (a) a nucleic acidenzyme dependent on an ion to produce a product; (b) a fluorophore; and(c) a photodetector.
 48. A composition comprising a nucleic acid enzymelinked to a fluorophore.
 49. A composition comprising a nucleic acidenzyme linked to a quencher.
 50. A composition comprising a nucleic acidenzyme, substrate, fluorophore, and quencher.
 51. A compositioncomprising a substrate for a nucleic acid enzyme linked to a quencher.52. A composition comprising a substrate for a nucleic acid enzymelinked to a fluorophore.